Cryptic peptides and method for their identification

ABSTRACT

The present invention relates to immunologically cryptic peptides; methods for their identification in individuals and populations and their use in diagnosis and therapy of pathological conditions such as asthma and allergy, and their use in screening for therapeutic activity. Such cryptic peptides are identified by a method which includes the steps of: 1) exposing T cells with the peptide in a primary challenge; 2) measuring the reactivity of T cells with the peptide in the primary challenge of step 1); 3) exposing pre-challenged T cells with the peptide in a secondary challenge, wherein the pre-challenged T cells are obtainable by exposign the T cells to the protein; and measuring the reactivity of the pre-challenged T cells with the peptide in the secondary challenge of step  3 ), and the peptide is a cryptic peptide if T-cell reactivity is observable in the secondary challenge but not in the primary challenge.

PRIORITY

This application is a National Stage application, under 35 U.S.C. §371,of International Application no. PCT/GB97/00783, filed Mar. 20, 1997,which claims priority to United Kingdom application nos. 9605904.3,filed Mar. 21, 1996, and 9608430.6, filed Apr. 24 1996.

The present invention relates to immunologically cryptic peptides;methods for their identification in individuals and populations andtheir use in diagnosis and therapy of pathological conditions such asasthma and allergy, and their use in screening for therapeutic activity.

The ability of the immune system to elicit a response to a particularmolecule depends critically upon its ability to recognise the presenceof an antigen. Classically, the term antigen was associated with theability of a molecule to be an antibody generator via induction ofB-cells. It is now known however that T cells also possess the abilityto recognise antigens. T-cell antigen recognition requires antigenpresenting cells (APCs) to present antigen fragments (peptides) on theircell surface in association with molecules of the majorhistocompatibility complex (MHC). T cells use their antigen specificreceptors (TCRs) to recognise the antigen fragments presented by theAPC. Such recognition acts as a trigger to the immune system to generatea range of responses to eradicate the antigen which has been recognised.

In order to trigger an individual T cell, a critical number of TCRs mustbe ligated by the peptide/MHC complex presented by the APC. A peptidewhich reaches the surface of the APC in sufficient numbers to do thiscan be termed “dominant” or “sub-dominant” depending upon its ability toinduce T cell activation relative to other triggering peptides. In thenormal course of events i.e. physiologically, a given protein willgenerate more than one peptide which is capable of triggering a T cellresponse. The term “dominant” would then be applied to the peptide thatinduces the most potent or most frequent response. In addition todominant and sub-dominant epitopes, there are potential T cell peptideepitopes within a given protein sequence (for which T cells arespecific), which do not reach the APC surface in sufficient numbers toinduce a response. In other words due to the mechanisms of antigenprocessing within the APC, certain peptides are processed and presentedto T-lymphocytes efficiently and therefore stimulate T-cell responseswhite others are poorly processed and presented to T-lymphocytes. Asthese latter peptides are not present upon the APC surface in sufficientnumbers to stimulate a potentially reactive T-lymphocyte, these peptideshave been referred to as “cryptic peptide epitopes”.

Cryptic peptide epitopes are present both in proteins normally presentin the body (self proteins) and in non-self (or foreign) proteins. Innormal physiology, T cells which have the capability of reacting with acryptic epitope cannot be detected in an in vitro primary stimulationassay (that is, T cells freshly isolated from the blood do not exhibitdemonstrable proliferation when cultured with the cryptic peptide). Incontrast other peptides which are efficiently processed and presented toT-lymphocytes by APC. can stimulate a proliferative response in primaryculture. These are the “dominant” and “sub-dominant” epitopes.

The term “atopic allergy” is applied to a group of allergiescharacterised by high concentrations of immunoglobulin E (IgE). Theyinclude allergic asthma, hay fever, perennial allergic rhinitis, someforms of urticaria (hives) and eczema, allergic conjunctivitis andcertain food allergies (particularly food anaphylaxis). The mechanismsof generation of the pathology of such atopic conditions involves notonly the synthesis of antigen/allergen specific IgE but also theaccompanying differentiation and growth of effector cells such as mastcells and eosinophils.

Allergic IgE-mediated diseases are currently treated by desensitizationprocedures that involve the periodic injection of allergen components orextracts. Desensitization treatments may induce an IgG response thatcompetes with IgE for allergen, or they may induce specific suppressor Tcells that block the synthesis of IgE directed against allergen. Thisform of treatment is not always effective and poses the risk ofprovoking serious side effects, particularly general anaphiylacticshock. This can be fatal unless recognised immediately and treated withadrenaline. A therapeutic treatment that would decrease or eliminate theunwanted allergic-immune response to a particular allergen, withoutaltering the immune reactivity to other foreign antigens or triggeringan allergic response itself would be of great benefit to allergicindividuals.

Sometimes the normal mechanisms whereby self and non-self areimmunologically distinguished may break down and an immune response maybe elicited against self-antigens present in normal body tissues. This“auto-immunity” generates pathological conditions such as autoimmunethyroiditis, rheumatoid arthritis and lupus erythematosus. Therapeuticregimes are generally limited to the use of anti-inflammatory orimmunosuppressive drugs which are relatively non-specific and have manyundesirable side-effects.

WO 92111859 describes a method of reducing immune response to anallergen in which a non-allergen derived, non-stimulating peptide whichbinds to specific MHC class 11 molecules of APCs is used to inhibitT-cell response to particular allergens.

WO 91/06571 purports to disclose peptides derived from human T-cellreactive feline protein which can be used in the diagnosis, treatment orprevention of cat allergy.

WO 94/24281 relates to peptides and modified peptides of the major housedust mite allergens. The modified peptides have the intent of reducingthe level of undesirable side effects associated with desensitizingtherapies.

G. F. Hoyne et. al. in Immunology 83 pp 190-195 (1994) examined housedust mite allergy using peptides made from cDNA encoding the majorallergen DerpI. They purport to show that peptides containing majorepitopes can induce oral tolerance in mice to the whole allergen andthat it is also possible to induce tolerance with other peptides.Cryptic peptides are suggested as playing a role in this process but nomethods are disclosed for their identification or therapeutic use.

None of the above disclosures makes any suggestion that cryptic peptidesmay play a role in the pathology of atopic conditions such as asthma orother allergic diseases. The present inventors have found a method foridentifying cryptic peptides and have observed that individuals withasthma or other allergy-based pathologies have T-lymphocyte populationswhich can be stimulated in primary culture by cryptic epitopes derivedfrom the allergen which causes the relevant pathology. As describedabove, T-lymphocytes isolated from a healthy individual would not beexpected to be stimulated in primary culture by a cryptic epitope.Likewise in autoimmune pathologies, a self peptide, normally cryptic tothe immune system becomes recognised and elicits an immune response.

Hence there is provided according to the invention, a method ofdetermining whether a peptide of a protein is a cryptic peptide, whichmethod includes the steps of: i) exposing T cells with the peptide in aprimary challenge; ii) measuring the reactivity of T cells with thepeptide in the primary challenge of Step i; iii) exposing pre-challengedT cells with the peptide in a secondary challenge, wherein thepre-challenged T cells are obtainable by exposing the T cells to theprotein; and measuring the reactivity of the pre-challenged T cells withthe peptide in the secondary challenge.

The prechallenge allows expression of not only dominant and sub-dominantepitopes on the APC surface, but also of any cryptic determinants. Thesubsequent peptide rechallenge of these cells reveals T-cell reactivityto the dominant, sub-dominant and cryptic epitopes. Primary challengewith peptides will elicit responses from only the normally expresseddominant and sub-dominant epitopes. Thus, peptides recognised followingwhole antigen primary challenge followed by peptide secondary challenge,but not after peptide primary challenge alone are by definition, crypticepitopes. The peptide is a cryptic peptide if T-cell reactivity isobservable in the secondary challenge above but not in the primarychallenge.

The present invention may be illustrated as follows. If peripheral bloodmononuclear cells (PBMC) which contain T cells are taken directly froman individual and incubated for a short period of time (e.g. 3-7 days)with a set of synthetic, overlapping peptides from a protein,proliferative responses will be seen to the peptides which T cellsrecognise in the normal course of events i.e. dominant and sub-dominantepitopes. If however, PBMC are first cultured for 1-2 weeks with a highdose of the whole molecule (or a cocktail of all the peptides),potential T-cell epitopes dominant, sub-dominant and cryptic will thenbe detected by subsequent “secondary” challenge with the peptides. Thisis because T-cell populations with any specificity for any of thepeptides will have been triggered and expanded (increased in numbers) asa result of the high dose “primary” challenge with whole antigen. Bychallenging PBMC with the peptides in a primary assay and also asecondary assay, cryptic epitopes can be identified within a population.Therefore any peptide which behaves as a dominant epitope in asthmaticsor individuals with a related pathology, but as a cryptic epitope in thenormal population can be detected using this method of primary andsecondary assay.

In preferred embodiments of the present invention the pre-challengedcells are obtained by exposing the T cells to protein or by exposing theT cells to protein in bulk culture.

The T cells may be obtained from a population comprising a number ofindividuals (e.g. >20) or from a single individual. If they are obtainedfrom a population then any peptide identified as cryptic in all healthyindividuals may be considered to be cryptic within the population ingeneral while use of T cells isolated from a single individual willidentify only peptides which are cryptic (or not) in that individual andmay not be cryptic in the population.

The steps of the method above may be carried out in the sequence asdescribed or in any alternative sequence known to the person skilled inthe art to be suitable to obtain an essentially equivalent result.

In a particularly preferred embodiment of the present invention, theprotein from which the peptides are derived is chosen from the listcomprising Fel dl (the feline skin and salivary gland allergen of thedomestic cat Felis domesticus—the amino acid sequence of which isdisclosed in WO 91/06571), Der p I, Der p II, Der fl or Der fll (themajor protein allergens from the house dust mite dermatophagoides—aminoacid sequences disclosed in WO 94/24281) and allergens present in any ofthe following: grass, tree and weed (including ragweed) pollens; fungiand molds; foods e.g. fish, shellfish, crab, lobster, peanuts, nuts, eggand milk; stinging insects e.g. bee, wasp and hornet and thechironomidae (non-biting midges); spiders and mites: mammals such asdog, horse, rat, guinea pig, mice and gerbil; latex; biologicaldetergent additives; drugs e.g. penicillins and other antibiotics andanaesthetic agents.

More particularly the insect protein from which the peptides may bederived is chosen from the list comprising: housefly, fruit fly, sheepblow fly, screw worm fly, grain weevel, silkworm, honeybee, non-bitingmidge larvae, bee moth larvae, mealworm, cockroach and larvae ofTenibrio molitor beetle. All these being insect allergens of particularrelevance to allergic problems arising in the workplace.

Particularly preferred peptides for use in the method of the inventioninclude those shown in FIGS. 1, 2 and 3. The method may also use thoseshown in any one of Sequence I.D. Nos 25 to 56. The invention alsoencompasses the use of these peptides as cryptic peptides.

A further aspect of the present invention is any peptide identified asbeing cryptic when screened by the method of the present invention.

Peptides retaining the ability to bind to MHC class 11 molecules mayhave up to around 40, preferably 31 residues. Thus, it will beappreciated that useful peptides may comprise a sequence as shown in anyone of Sequence I.D. Nos 2 to 10 and 12 to 56. Thus, in one preferredexample a 14 mer contiguous sequence forms part of a larger peptide,preferably one up to about 31 residues. In this example the 14 mer formsabout 45% of the larger peptide (or polypeptide). Preferably thesequence forms about 50% or more, more preferably about 60% or more,even more preferably about 70% or more, or about 80% or more of thelarger peptide. In a specially preferred embodiment the sequence formsabout 90% or about 95% or more of the larger sequence.

Yet further aspects of the present invention include a peptide of thepresent invention, preferably a cryptic peptide when screened by themethod of the present invention for use as a medicament or as adiagnostic; the use of a peptide of the present invention, preferably acryptic peptide when screened by the method of the present invention, inthe preparation of a medicament for the treatment of atopic conditionssuch as asthma; the use of a peptide of the present invention,preferably a cryptic peptide when screened by the method of the presentinvention, in the preparation of a diagnostic for the diagnosis ofatopic conditions such as asthma; a method of preparing a medicament ordiagnostic comprising mixing a peptide of the present invention,preferably a cryptic peptide when screened by the method of the presentinvention, with a suitable carrier, diluent or excipient; theformulations prepared from such uses and methods; a method of therapyand/or diagnosis practised on the human body using a peptide of thepresent invention, preferably a cryptic peptide.

Whilst it may be possible for the peptides of the present invention tobe administered as the raw peptide, it is preferable to present them asa pharmaceutical formulation. According to a further aspect, the presentinvention provides a pharmaceutical formulation comprising a crypticpeptide together with one or more pharmaceutically acceptable carrierstherefor and optionally one or more other therapeutic ingredients. Thecarrier(s) must be ‘acceptable’ in the sense of being compatible withthe other ingredients of the formulation and not deleterious to therecipient thereof.

The formulations include those suitable for oral (particularly inhaled),parenteral (including subcutaneous, transdermal, intradermal,intramuscular and intravenous and rectal) administration although themost suitable route may depend upon for example the condition anddisorder of the recipient. The formulations may conveniently bepresented in unit dosage form and may be prepared by any of the methodswell known in the art of pharmacy. All methods include the step ofbringing into association a compound of the present invention as hereindefined or a pharmacologically acceptable salt or solvate thereof(“active ingredient”) with the carrier which constitutes one or moreaccessory ingredients.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste. Formulations for inhalationmay be presented in any of the ways known to be effective e.g. metereddoes inhalers.

Formulations for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water-for-injection, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets of the kindpreviously described.

Formulations for rectal administration may be presented as a suppositorywith the usual carriers such as cocoa butter or polyethylene glycol.

Preferred unit dosage formulations are those containing an effectivedose, as hereinbelow recited, or an appropriate fraction thereof, of theactive ingredient.

The compounds of the invention may typically be administered orally orvia injection at a dose of from 0.001 to 1 mg/kg per day.

The present invention also provides diagnostic kits which comprisecryptic peptides of the present invention. Each kit consists of amicroculture plate containing lyophilized peptides corresponding to thecryptic peptide(s) identified by the method of the invention. Eachpeptide may be present in a minimum of three wells (for statisticalevaluation). More replicates may be used depending on the number ofpeptides to be screened. The use of lyophilized peptides allows the kitto be stored at room temperature for several months before use.

According to a preferred embodiment for carrying out the assay,peripheral blood mononuclear cells (PBMC) may be isolated from patientblood by standard methods. Approximately 20 ml of blood is required.PBMC is added to the wells of the microculture plate in a volume of 200μl of culture medium containing 10⁵ cells. After 6 days of culture in ahumidified incubator at 37° C. gased with 5% CO₂ in air, an isotopiclabel such as ³H-thymidine or, a non-isotopic label such asbromo-deoxyuridine is added to each well for a prescribed period(approx. 12-24 hours). Cell proliferation to the peptides present may beassayed in any suitable way, e.g. DNA synthetic analysis, liquidscintillation spectroscopy or colourimetrically.

The measurement of cellular reactivity to peptide challenge according tothe present invention is preferably carried out by measurement ofcellular proliferation as discussed above. However such reactivity mayalso be measured by determination of other cellular responses e.g. assayfor the release of soluble mediators such as cytokines and chemokineswhich may demonstrate a release profile indicative of allergicresponses.

Cryptic peptides of the present invention which are to be used intherapy may be subject to point mutation in order to avoid certainundesirable side effect of peptide immunotherapy and/or to improve theireffects.

Peptide immunotherapy involves the administration of peptides which willdisable specific T cells. Data from the trial of Allervax Cat [J.Allergy Clin. Immunol. Jan 1996 Abstract 815] shows that in somepatients, a late-phase response to the administered peptide occurs. Thisresponse is essentially an asthma attack and represents a seriousside-effect of this kind of therapy. By use of “altered peptide ligands”the same therapeutic goals can be achieved i.e., functional eliminationof reactive T cells, but without the development of a late-phaseresponse. Having identified a cryptic peptide for therapeutic use, aseries of a point mutations may be made to residues in order to generatea panel of closely related peptides. These may be screened for thereability to a) bind to the appropriate MHC molecule with a similaraffinity to the original peptide and b) anergise or kill T cellsspecific for the original peptide. Such altered or synthetic peptidesare also encompassed within the present invention.

Particularly preferred synthetic peptides, according to the presentinvention and for use according to the present invention, include thoseshown in FIG. 3. Such peptides may be used according to the presentinvention either individually or in any pairwise or multiple combinationthereof.

The pre-challenged T cells may be generated as described above or in onealternative they may be generated by exposure of the T cells to bulkculture or to multiple peptides derived from the protein of interest.

In a particularly preferred embodiment according to the presentinvention, the method for determining whether a peptide of a protein isa cryptic peptide may be carried out as follows. T cells are isolatedfrom peripheral blood using established techniques [Cellular ImmunologyLabFax., P. J. Delves (Ed), 1994, Tissue and Cell Culture, Chapter 4,pp. 45. Blackwell Scientific Publications]. A proportion of the cellsare cultured in vitro for 6-12 days with the protein of interest. Therest of the cells are aliquoted into cultures and incubated with thepeptide of interest derived from the protein. After 3-6 days a label isadded to the culture which enables the quantity of DNA synthesis withinthe cultures to be quantified sometime later (e.g. 8-16 hours later). Byanalysing the quantity of DNA in the cultures compared to a controlculture containing no peptide, it is possible to identify peptidesequences from the protein which stimulate T cells in this “primary”challenge. The group of cells cultured for 6-12 days with whole proteinare collected at the end of this culture and washed several times inculture medium. However, as these cells were cultured with whole proteinfirst i.e. were “pre-challenged” the subsequent peptide challenges arereferred to as “secondary” challenges. Subsequent analysis of quantitiesof DNA synthesis in secondary challenges will identify all peptidesderived from the protein which can potentially be recognised by T cellswithin an individual.

In summary, the primary cultures identify the peptides which arerecognised by T-lymphocytes from individuals with disease or withoutdisease and the secondary cultures identify all peptides to which Tcells can react in individuals with disease or without disease. Epitopeswhich are cryptic at the population level will be recognised in primaryculture by individuals with disease but not by individuals withoutdisease.

T cells according to the present invention may be taken to be anypreparation of mononuclear cells obtained from one or more individualscontaining T-lymphocytes at a purity sufficient to be able to detectreactivity in a peptide challenge. All technical processes describedabove may alternatively be carried out using another process known tothe person skilled in the art to be able to achieve the desired purposeof that process.

In a further embodiment of the present invention, there is provided theuse of cryptic peptides in a compound screen for the identification ofcompounds possessing therapeutic activity particularly in respect ofatopic conditions and more particulary in respect of asthma. In thisfurther embodiment of the present invention the cryptic peptides arepreferably those identified using the method of the present invention.

According to this further embodiment of the present invention there isprovided both a screening process for the identification of therapeuticcompounds and a kit adapted to put such screening process into effect.The screening process of the present invention is preferably carried outin vitro, but may also be carried out in vivo. In a preferredembodiment, the screening process of the present invention may becarried out in vitro as follows. Isolated peripheral blood mononuclearcells or cultured T lymphocytes are cultured in vitro withconcentrations of one or more cryptic peptides, preferably previouslydetermined as being optimal for the induction of proliferative and/orother responses in these cells. To certain of the culture wells areadded, compounds which are to be screened for their potential todiminish the proliferative response of these cells to the crypticpeptide. Such compounds are preferably added to cultures over a broadrange of concentrations. For example, the compound may be added to thecultures at 10-fold dilutions over the range 10⁻¹²M to 10⁻²M in order toidentify the dose providing the largest diminution in the proliferativeresponse of the cells. Cellular proliferation may be measured using anytechnique well known in the art, for example cells may be labelled witha compound such as tritiated thymidine which will enable quantificationof DNA synthesis and thus cellular proliferation. Compounds capable ofdiminishing the cellular proliferation induced by the cryptic peptidemay be identified as a therapeutic in respect of a medical conditionassociated with that cryptic peptide. When carried out in vivo, thescreening process comprises the administration preferably to a humanmammal, of a cryptic peptide to induce a condition associated with thatcryptic peptide and the administration, preferably subsequently, of acompound to be screened. A compound capable of diminishing symptomsassociated with the induced condition may be identified as a therapeuticin respect of that condition.

Preferably the screen whether in vitro or in vivo comprises the use ofcryptic peptides associated with atopic conditions such as asthma andmost preferably the use of one or more of the FC1P peptides identifiedherein.

Non-limiting examples of the invention will now be described withreference to the accompanying Figures, in which:

FIG. 1: shows the native sequence of chain 1 of the major cat allergenFel d I in single letter amino acid code; and nine peptides derivedtherefrom. The identifiers for the sequences depicted are as follows:Native sequence Chain 1 (SEQ ID NO:1); Peptide 1 (SEQ ID NO:2); Peptide2 (SEQ ID NO:3); Peptide 3 (SEQ ID NO: 4); Peptide 4 (SEQ ID NO:5);Peptide 5 (SEQ ID NO:6); Peptide 6 (SEQ ID NO:7); Peptide 7 (SEQ IDNO:8); Peptide 8 (SEQ ID NO:9); Peptide 9 (SEQ ID NO:10)

FIG. 2: shows the native sequence of chain 2 of the major cat allergenFel d I in single letter amino acid code: and eleven peptides derivedtherefrom. The identifiers for the sequences depicted are as follows:Native sequence Chain 2 (SEQ ID NO: 11); Peptide 2.1 (SEQ ID NO: 12);Peptide 2.2 (SEQ ID NO: 13); Peptide 2.2 (SEQ ID NO: 14); Peptide 2.3(SEQ ID NO:15); Peptide 2.4 (SEQ ID NO:16); Peptide 2.5 (SEQ ID NO:17);Peptide 2.6 (SEQ ID NO:18); Peptide 2.7 (SEQ ID NO:18); Peptide 2.8 (SEQID NO:19); Peptide 2.9 (SEQ ID NO:20; Peptide 2.10 (SEQ ID NO:21);Peptide 2.11 (SEQ ID NO:22).

FIG. 3: shows three synthetic peptides derived from the sequence of Feld I. The three peptides are collectively referred to as FC1P. Theidentifiers for the sequences depicted are as follows: the top sequence(SEQ ID NO:23); the middle sequence (SEQ ID NO:5); the bottom sequence(SEQ ID NO:24).

FIG. 4: shows the results of Example 2 in terms of levels of cellularproliferation (expressed as Δ cpm) in response to primary and secondarychallenge of PBMCs with FC1P, as described of PBMCs in the Example.

FIG. 5: shows the results of Example 3 in terms of levels of cellularproliferation (expressed as Δ cpm) in response to primary challenge ofPBMCs with FC1P, as described in the Example.

FIG. 6: shows the results of Example 4 in terms of the lung function ofan asthmatic (expressed as FEV₁ in litres) over time, as a consequenceof the factors described in the Example.

FIG. 7: shows the results of Example 5 in terms of inhibition of T cellproliferation (measured by tritiated thymidine incorporation) to FC1 Ppeptides by dexamethazone and fluticazone.

EXAMPLE 1

The method for determining whether a peptide of a protein is a crypticpeptide may be carried out as follows.

1—T cells are isolated from peripheral blood,

2—a proportion of the cells are cultured in vitro for 6-12 days with theprotein of interest.

3—the rest of the cells are aliquoted into cultures and incubated withthe peptide of interest derived from the protein,

4—after 3-6 days a label is added to the culture, enabling the quantityof DNA synthesis within the cultures to be quantified.

5—analysis of the quantity of DNA synthesis in the cultures compared toa control culture containing no peptide, this identifies the peptidesequences from the protein which stimulate T cells in this “primary”challenge,

6—the group of cells cultured for 6-12 days with whole protein arecollected at the end of this culture and washed several times in culturemedium,

7—analysis of quantities of DNA synthesis in secondary challenges willidentify all peptides derived from the protein which can be recognisedby T cells within an individual.

The cryptic peptides identified by this method are those stimulatingT-cell reactivity in the secondary challenge but not in the primarychallenge.

EXAMPLE 2

Peripheral blood mononuclear cells were isolated by density gradientcentrifugation. A proportion of the cells were challenged with peptidesin primary culture and the remaining cells cultured for 10 days with 100μg/ml cat dander extract (containing Fel d I). Subsequently, cellscultured with cat dander were collected, washed in culture medium andthen cultured for a further three days with the three peptides (at aconcentration of 36 μg/ml) which together constitute FC1P (as shown inthe FIG. 3 herein) and equal numbers of irradiated autologous feedercells. Cellular proliferation (expressed as Δ cpm) was quantified bymeasurement of incorporation of tritiated thymidine into newlysynthesised DNA, and is illustrated in FIG. 4.

EXAMPLE 3

Freshly isolated peripheral blood mononuclear cells were cultured at 10⁵cells per well for 6 days prior to labelling with tritiated thymidinefor measurement of cellular proliferation. Cat allergic asthmatics (CAA)demonstrate greater primary proliferative responses to FC1P (36 μm/ml)than do non-cat allergic asthmatics (NCA). The results are shown in FIG.5.

EXAMPLE 4

40 μg of highly purified FC1P peptides was injected intradermally intoeach forearm of a volunteer cat-allergic asthmatic. This induced anisolated late-phase asrimatic reaction approximately three hours afteradministration of FC1P. Lung function (quantified as FEV₁) fell andremained low over the next several hours. The drop in FEV₁ was reversedby the administration of β₂ agonist and inhaled corticosteroid at 8hours. The results of this are shown diagrammatically in FIG. 6.

EXAMPLE 5

Freshly isolated T lymphocytes were cultured in vitro withconcentrations of FC1P peptides, previously determined as being optimalfor the induction of proliferative and/or other responses in thesecells. To certain of the culture wells were added, theglucocorticosteroid Dexamethazone or fluticazone, these were added tocultures at dilutions of 10⁻⁶M or 10⁻⁹M. After culture, cells werelabelled with tritiated thymidine to enable quantification of DNAsynthesis and thus cellular proliferation. The results are shown in FIG.7.

56 1 70 PRT Felis domesticus 1 Glu Ile Cys Pro Ala Val Lys Arg Asp ValAsp Leu Phe Leu Thr Gly 1 5 10 15 Thr Pro Asp Glu Tyr Val Glu Gln ValAla Gln Tyr Lys Ala Leu Pro 20 25 30 Val Val Leu Glu Asn Ala Arg Ile LeuLys Asn Cys Val Asp Ala Lys 35 40 45 Met Thr Glu Glu Asp Lys Glu Asn AlaLeu Ser Leu Leu Asp Lys Ile 50 55 60 Tyr Thr Ser Pro Leu Cys 65 70 2 15PRT Felis domesticus 2 Glu Ile Cys Pro Ala Val Lys Arg Asp Val Asp LeuPhe Leu Thr 1 5 10 15 3 16 PRT Felis domesticus 3 Arg Asp Val Asp LeuPhe Leu Thr Gly Thr Pro Asp Glu Tyr Val Glu 1 5 10 15 4 16 PRT Felisdomesticus 4 Gly Thr Pro Asp Glu Tyr Val Glu Gln Val Ala Gln Tyr Lys AlaLeu 1 5 10 15 5 16 PRT Felis domesticus 5 Glu Gln Val Ala Gln Tyr LysAla Leu Pro Val Val Leu Glu Asn Ala 1 5 10 15 6 15 PRT Felis domesticus6 Tyr Lys Ala Leu Pro Val Val Leu Glu Asn Ala Arg Ile Leu Lys 1 5 10 157 16 PRT Felis domesticus 7 Pro Val Val Leu Glu Asn Ala Arg Ile Leu LysAsn Cys Val Asp Ala 1 5 10 15 8 17 PRT Felis domesticus 8 Arg Ile LeuLys Asn Cys Val Asp Ala Lys Met Thr Glu Glu Asp Lys 1 5 10 15 Glu 9 16PRT Felis domesticus 9 Lys Met Thr Glu Glu Asp Lys Glu Asn Ala Leu SerLeu Leu Asp Lys 1 5 10 15 10 15 PRT Felis domesticus 10 Asn Ala Leu SerLeu Leu Asp Lys Ile Tyr Thr Ser Pro Leu Cys 1 5 10 15 11 92 PRT Felisdomesticus 11 Val Lys Met Ala Glu Thr Cys Pro Ile Phe Tyr Asp Val PhePhe Ala 1 5 10 15 Val Ala Asn Gly Asn Glu Leu Leu Leu Lys Leu Ser LeuThr Lys Val 20 25 30 Asn Ala Thr Glu Pro Glu Arg Thr Ala Met Lys Lys IleGln Asp Cys 35 40 45 Tyr Val Glu Asn Gly Leu Ile Ser Arg Val Leu Asp GlyLeu Val Met 50 55 60 Thr Thr Ile Ser Ser Ser Lys Asp Cys Met Gly Glu AlaVal Gln Asn 65 70 75 80 Thr Val Glu Asp Leu Lys Leu Asn Thr Leu Gly Arg85 90 12 16 PRT Felis domesticus 12 Val Lys Met Ala Glu Thr Cys Pro IlePhe Tyr Asp Val Phe Phe Ala 1 5 10 15 13 14 PRT Felis domesticus 13 IlePhe Tyr Asp Val Phe Phe Ala Val Ala Asn Gly Asn Glu 1 5 10 14 16 PRTFelis domesticus 14 Val Ala Asn Gly Asn Glu Leu Leu Leu Lys Leu Ser LeuThr Lys 1 5 10 15 Val 15 17 PRT Felis domesticus 15 Glu Leu Leu Leu LysLeu Ser Leu Thr Lys Val Asn Ala Thr Glu Pro 1 5 10 15 Glu 16 15 PRTFelis domesticus 16 Asn Ala Thr Glu Pro Glu Arg Thr Ala Met Lys Lys IleGln Asp 1 5 10 15 17 16 PRT Felis domesticus 17 Arg Thr Ala Met Lys LysIle Gln Asp Cys Tyr Val Glu Asn Gly Leu 1 5 10 15 18 16 PRT Felisdomesticus 18 Cys Tyr Val Glu Asn Gly Leu Ile Ser Arg Val Leu Asp GlyLeu Val 1 5 10 15 19 16 PRT Felis domesticus 19 Ile Ser Arg Val Asp GlyLeu Val Met Ile Thr Thr Ile Ser Ser Ser 1 5 10 15 20 15 PRT Felisdomesticus 20 Met Thr Thr Ile Ser Ser Ser Lys Asp Cys Met Gly Glu AlaVal 1 5 10 15 21 16 PRT Felis domesticus 21 Lys Asp Cys Met Gly Glu AlaVal Gln Asn Thr Val Glu Asp Leu Lys 1 5 10 15 22 14 PRT Felis domesticus22 Gln Asn Thr Val Glu Asp Leu Lys Leu Asn Thr Leu Gly Arg 1 5 10 23 17PRT Felis domesticus 23 Leu Phe Leu Thr Gly Thr Pro Asp Glu Tyr Val GluGln Val Ala Gln 1 5 10 15 Tyr 24 17 PRT Felis domesticus 24 Lys Ala LeuPro Val Val Leu Glu Asn Ala Arg Ile Leu Lys Asn Cys 1 5 10 15 Val 25 14PRT Felis domesticus 25 Glu Ile Cys Pro Ala Val Lys Arg Asp Val Asp LeuPhe Leu 1 5 10 26 14 PRT Artificial Sequence Description of ArtificialSequenceCryptic peptide derived from allergens 26 Ile Cys Pro Ala ValLys Arg Asp Val Asp Leu Phe Leu Thr 1 5 10 27 14 PRT Artificial SequenceDescription of Artificial SequenceCryptic peptide derived from allergens27 Cys Pro Ala Val Lys Arg Asp Val Asp Leu Phe Leu Thr Gly 1 5 10 28 14PRT Artificial Sequence Description of Artificial SequenceCrypticpeptide derived from allergens 28 Pro Ala Val Lys Arg Asp Val Asp LeuPhe Leu Thr Gly Thr 1 5 10 29 14 PRT Artificial Sequence Description ofArtificial SequenceCryptic peptide derived from allergens 29 Ala Val LysArg Asp Val Asp Leu Phe Leu Thr Gly Thr Pro 1 5 10 30 14 PRT ArtificialSequence Description of Artificial SequenceCryptic peptide derived fromallergens 30 Val Lys Arg Asp Val Asp Leu Phe Leu Thr Gly Thr Pro Asp 1 510 31 14 PRT Artificial Sequence Description of ArtificialSequenceCryptic peptide derived from allergens 31 Lys Arg Asp Val AspLeu Phe Leu Thr Gly Thr Pro Asp Glu 1 5 10 32 14 PRT Artificial SequenceDescription of Artificial SequenceCryptic peptide derived from allergens32 Arg Asp Val Asp Leu Phe Leu Thr Gly Thr Pro Asp Glu Tyr 1 5 10 33 14PRT Artificial Sequence Description of Artificial SequenceCrypticpeptide derived from allergens 33 Asp Val Asp Leu Phe Leu Thr Gly ThrPro Asp Glu Tyr Val 1 5 10 34 14 PRT Artificial Sequence Description ofArtificial SequenceCryptic peptide derived from allergens 34 Val Asp LeuPhe Leu Thr Gly Thr Pro Asp Glu Tyr Val Glu 1 5 10 35 14 PRT ArtificialSequence Description of Artificial SequenceCryptic peptide derived fromallergens 35 Asp Leu Phe Leu Thr Gly Thr Pro Asp Glu Tyr Val Glu Gln 1 510 36 14 PRT Artificial Sequence Description of ArtificialSequenceCryptic peptide derived from allergens 36 Leu Phe Leu Thr GlyThr Pro Asp Glu Tyr Val Glu Gln Val 1 5 10 37 14 PRT Artificial SequenceDescription of Artificial SequenceCryptic peptide derived from allergens37 Phe Leu Thr Gly Thr Pro Asp Glu Tyr Val Glu Gln Val Ala 1 5 10 38 14PRT Artificial Sequence Description of Artificial SequenceCrypticpeptide derived from allergens 38 Leu Thr Gly Thr Pro Asp Glu Tyr ValGlu Gln Val Ala Gln 1 5 10 39 14 PRT Artificial Sequence Description ofArtificial SequenceCryptic peptide derived from allergens 39 Thr Gly ThrPro Asp Glu Tyr Val Glu Gln Val Ala Gln Tyr 1 5 10 40 14 PRT ArtificialSequence Description of Artificial SequenceCryptic peptide derived fromallergens 40 Gly Thr Pro Asp Glu Tyr Val Glu Gln Val Ala Gln Tyr Lys 1 510 41 14 PRT Artificial Sequence Description of ArtificialSequenceCryptic peptide derived from allergens 41 Thr Pro Asp Glu TyrVal Glu Gln Val Ala Gln Tyr Lys Ala 1 5 10 42 14 PRT Artificial SequenceDescription of Artificial SequenceCryptic peptide derived from allergens42 Pro Asp Glu Tyr Val Glu Gln Val Ala Gln Tyr Lys Ala Leu 1 5 10 43 14PRT Artificial Sequence Description of Artificial SequenceCrypticpeptide derived from allergens 43 Asp Glu Tyr Val Glu Gln Val Ala GlnTyr Lys Ala Leu Pro 1 5 10 44 14 PRT Artificial Sequence Description ofArtificial SequenceCryptic peptide derived from allergens 44 Glu Tyr ValGlu Gln Val Ala Gln Tyr Lys Ala Leu Pro Val 1 5 10 45 14 PRT ArtificialSequence Description of Artificial SequenceCryptic peptide derived fromallergens 45 Tyr Val Glu Gln Val Ala Gln Tyr Lys Ala Leu Pro Val Val 1 510 46 14 PRT Artificial Sequence Description of ArtificialSequenceCryptic peptide derived from allergens 46 Val Glu Gln Val AlaGln Tyr Lys Ala Leu Pro Val Val Leu 1 5 10 47 14 PRT Artificial SequenceDescription of Artificial SequenceCryptic peptide derived from allergens47 Glu Gln Val Ala Gln Tyr Lys Ala Leu Pro Val Val Leu Glu 1 5 10 48 14PRT Artificial Sequence Description of Artificial SequenceCrypticpeptide derived from allergens 48 Gln Val Ala Gln Tyr Lys Ala Leu ProVal Val Leu Glu Asn 1 5 10 49 14 PRT Artificial Sequence Description ofArtificial SequenceCryptic peptide derived from allergens 49 Val Ala GlnTyr Lys Ala Leu Pro Val Val Leu Glu Asn Ala 1 5 10 50 14 PRT ArtificialSequence Description of Artificial SequenceCryptic peptide derived fromallergens 50 Ala Gln Tyr Lys Ala Leu Pro Val Val Leu Glu Asn Ala Arg 1 510 51 14 PRT Artificial Sequence Description of ArtificialSequenceCryptic peptide derived from allergens 51 Gln Tyr Lys Ala LeuPro Val Val Leu Glu Asn Ala Arg Ile 1 5 10 52 14 PRT Artificial SequenceDescription of Artificial SequenceCryptic peptide derived from allergens52 Tyr Lys Ala Leu Pro Val Val Leu Glu Asn Ala Arg Ile Leu 1 5 10 53 14PRT Artificial Sequence Description of Artificial SequenceCrypticpeptide derived from allergens 53 Lys Ala Leu Pro Val Val Leu Glu AsnAla Arg Ile Leu Lys 1 5 10 54 14 PRT Artificial Sequence Description ofArtificial SequenceCryptic peptide derived from allergens 54 Ala Leu ProVal Val Leu Glu Asn Ala Arg Ile Leu Lys Asn 1 5 10 55 14 PRT ArtificialSequence Description of Artificial SequenceCryptic peptide derived fromallergens 55 Leu Pro Val Val Leu Glu Asn Ala Arg Ile Leu Lys Asn Cys 1 510 56 14 PRT Artificial Sequence Description of ArtificialSequenceCryptic peptide derived from allergens 56 Pro Val Val Leu GluAsn Ala Arg Ile Leu Lys Asn Cys Val 1 5 10

What is claimed is:
 1. A peptide consisting of an amino acid sequenceselected from the group consisting of SEQ ID Nos. 2-4, 6-9, 12-21,25-27, 29-41, 43-46, 48-52, and 54-56.
 2. A peptide consisting of SEQ IDNO.
 5. 3. A peptide consisting of SEQ ID NO.
 23. 4. A peptide consistingof SEQ ID NO.
 24. 5. A composition comprising: at least two peptides,each peptide consisting of an amino acid sequence selected from thegroup consisting of SEQ ID NO. 5, SEQ ID NO. 23, and SEQ ID NO. 24,wherein each peptide comprises a different amino acid sequence.
 6. Acomposition comprising: a peptide comprising an amino acid sequenceaccording to SEQ ID NO. 5; a peptide comprising an amino acid sequenceaccording to SEQ ID NO. 23; a peptide comprising an amino acid sequenceaccording to SEQ ID NO. 24; and a suitable carrier, diluent, orexcipient; wherein the amino acid sequences of said SEQ ID Nos 5, 23,and 24 are different.